In bio-cleanrooms or on production lines of pharmaceutical and food industries, monitoring of microorganisms is required for environmental control, and thus airborne microbes, falling microbes, and adherent microbes are counted. A measuring method is defined in ISO 14698-1 by the International Organization for Standardization for biocontamination control in cleanrooms. The cleanliness measured by the method is classified by grade. As methods of measuring airborne microorganisms, those utilizing an airborne-microbe sampler (refer to Patent Document 1) that sucks free-fall airborne microbes and a constant amount of air are generally used. In these methodologies, microbes are collected on an agar plate culture medium within a certain amount of time, and representation is made based on the number of colonies developing after cultivation. The above agar culture medium is cultured for two to seven days in an isothermal unit, and the number of colonies developed is visually counted. Then, the numbers of colonies on the culture media are averaged to obtain the number of airborne microbes. Note that, in facilities for manufacturing aseptic drugs and cell processing centers (CPCs) for producing cells where a high degree of cleanliness is required in cleanrooms, in the above-describe grade classification, the control has to be made with always maintaining Grade A or Grade B. This count for a number of particulates in the air of ≦3,530/m3 and a number of microbes of ≦10 CFU (Colony Forming Unit)/m3 (Grade A (inside a safety cabinet): ≦1 CFU/m3; and Grade B (a workplace near a safety cabinet): ≦10 CFU/m3).
Meanwhile, in another method (ATP method), luciferase and luciferin, which are chemiluminescence reagents, are added to ATP (adenosine triphosphate) contained in microorganisms for measuring bioluminescence. Dusts including microbes are retrieved by an airborne-microbe sampler onto an agar culture medium maintained in a dish, and are then spread to solutions or the like, thereby extracting ATP from the microbes by reagent reaction. The extracted ATP is measured by a bioluminescence method, and the amount of ATP is calculated from the obtained emission intensity. Since the amount of ATP and the number of microbes has a proportional relation, the number of airborne microbes can be calculated from the amount of ATP.
The following is a general measurement flow for airborne microbes using the ATP method.
1. Harvesting microbes by an airborne-microbe sampler;
2. Retrieving gathered microbes and spreading them to solutions or the like;
3. Eliminating dead microbes in a microbe liquid and eliminating exogenous ATP not derived from microbes;
4. Extracting ATP in viable microbes; and
5. Bioluminescence, measurement, and counting by adding a chemiluminescence reagent.
Regarding the above measurement flow 1, an impactor-type sampler in a porous nozzle form is used, and dusts included in 1 m3 of air are retrieved for 10 to 20 minutes onto a plate culture medium in a petri dish (refer to Non-Patent Document 1). The ratio of retrieving microbes is equal to or greater than 90%. In cleanrooms and safety cabinets with a high degree of cleanliness, several microbes are gathered by taking time of about 20 minutes.
Regarding the above measurement flow 2,                the microbes gathered onto the plate culture medium through collection are peeled off from the surface, and are retrieved into a separately-prepared container as a liquid sample. To totally retrieve microbes, retrieval of the microbes captured on the culture medium has to be reliably performed, and the skills of the operator and selection of a solution are important. Also, here, if the tool for retrieval itself is contaminated, correct counting cannot be performed.        
Regarding the above measurement flows 3 and 4,                along with an advance of development of reagents (refer to Non-Patent Document 3), it has become possible to easily eliminate dead microbes and exogenous ATP and extract ATP in microbes. An ATP eliminating reagent containing apyrase and adenosine phosphate deaminase as principal components is added to a liquid sample to be measured for reaction for 30 minutes, and then an ATP extraction reagent containing a surface-active agent as a principal component is added for reaction for 30 to 60 seconds. In this manner, a preparation for measuring only ATP derived from viable microbes is ready.        
Regarding the above measurement flow 5,                biochemical emission by adding a chemiluminescence reagent to the extracted liquid after ATP derived from viable microbes is extracted or by retrieving the extracted liquid after ATP derived from viable microbes is extracted and adding and mixing the extracted liquid with a chemiluminescence reagent is detected by a photodetector. Since the number of microbes is proportional to the amount of ATP, the number of microbes is calculated from the amount of ATP chemiluminescence.        
In an ATP chemiluminescence assay, to measure ATP chemiluminescence at a high degree of sensitivity and a high accuracy, it is important to use a highly-sensitive detector and achieving a high degree of light gathering with an optical arrangement of the detector and a chemiluminescence reaction field. Furthermore, it is important to achieve a light-shielding mechanism for suppressing the entrance of stray light as much as possible because the accuracy of chemiluminescence measurement is decreased when light coming from the outside of the apparatus or coming from things other than the chemiluminescent substance, which is called stray light, enters the inside of the apparatus. According to FIG. 2 of Non-Patent Document 1 (paper), a lower limit of detecting ATP is 1×10−17 mol (10 amol), which corresponds to a degree of sensitivity on the order of ten microbes in terms of the number of microbes. When airborne microbes are taken as a target, a detection sensitivity allowing one microbe to be detected is required. Therefore, in Non-Patent Document 1, cultivation at 35° C. for six hours is often inserted between the above measurement flows 1 and 2 (FIG. 9 of Non-Patent Document 1 (paper)).
In recent years, the amount of ATP can be measured from 1 amol with a bioluminescence detection system in which a dispenser and a detector are placed in a light-shielded space in a same device (FIG. 3 of Non-Patent Document 2 (paper)).
As to highly sensitive detectors, conventionally, a photomultiplier is used as a photodetector of a microorganism counting device including a luminometer for ATP measurement or a luminometer using ATP chemiluminescence. When using more highly sensitive detectors, photocounting is adopted in which a signal of the photomultiplier is subjected to digital processing. Next, as to an optical arrangement, since light intensity is attenuated by the square of a distance from a chemiluminescence-emitting point, it is said to be preferable to make a sample container containing chemiluminescent substance closer to a light-receiving surface. Also, since light from the chemiluminescence-emitting point is scattered in a spherical form, an optical arrangement for efficient retrieval onto the light-receiving surface is important. Note that, while light retrieval efficiency is often defined with a solid angle, in this manner, in order to achieve a higher sensitivity, it is important to make the light-receiving surface closer to the container and to prepare a large light-receiving surface with respect to a light-emitting area. It is also effective to surround a container holder with a mirror-surface member so as to forcefully reflect light from a mirror surface and to guide the light to the light-receiving surface.
Note that the order of the measurement flows 2, 3, and 4 is random. In an embodiment, the flows 3 and 4 are performed first, and then an ATP-extracted microbe liquid is retrieved in the flow 2.
Also, if falling microbes and adherent microbes are collected by a dedicated carrier in the measurement flow 1, the falling microbes and the adherent microbes can be measured in the same process from the measurement flow 2 onward.
Patent Documents
    Patent Document 1: Japanese Patent Application Laid-Open Publication No. 2000-300246    Patent Document 2: Japanese Patent Application Laid-Open Publication No. 2000-314738    Patent Document 3: Japanese Patent Application Laid-Open Publication No. 7-83831Non-Patent Documents    Non-Patent Document 1: Journal of Japan Air Cleaning Association, Vol. 38, No. 5, pp. 21-26, 2000    Non-Patent Document 2: Proceeding of 26th Annual Technological Meeting on Air Cleaning and Contamination Control, pp. 240-242, 2008    Non-Patent Document 3: Nippon Nogeikagaku Kaishi Vol. 78, No. 7, pp. 630-635, 2004